The American Journal of Pathology, Vol. 185, No. 1, January 2015

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IMMUNOPATHOLOGY AND INFECTIOUS DISEASES

Tumor Necrosis Factor a Decreases
Glucagon-Like Peptide-2 Expression by
Up-Regulating G-ProteineCoupled Receptor
120 in Crohn Disease
Takuya Tsukahara,* Kenji Watanabe,* Toshio Watanabe,* Hirokazu Yamagami,* Mitsue Sogawa,* Tetsuya Tanigawa,*
Masatsugu Shiba,* Kazunari Tominaga,* Yasuhiro Fujiwara,* Kiyoshi Maeda,y Kosei Hirakawa,y and Tetsuo Arakawa*
From the Departments of Gastroenterology* and Surgical Oncology,y Osaka City University Graduate School of Medicine, Osaka, Japan
Accepted for publication
September 24, 2014.
Address correspondence toToshio Watanabe, M.D., Ph.D.,
Department of Gastroenterology, Osaka City University
Graduate School of Medicine,
1-4-3 Asahi-machi Abeno-ku,
Osaka, Japan. E-mail:watanabet@med.osaka-cu.ac.jp.

Glucagon-like peptide (GLP)-2, secreted by L cells in the small intestine, has anti-inﬂammatory effects
in the gastrointestinal tract. A GLP-2 analogue has been an effective treatment for Crohn disease (CD).
G-proteinecoupled receptor (GPR) 40 and GPR120 are probably involved in GLP-2 production, themechanisms of which remain unclear. In our experiments, normal ileal mucosa expressed GPR40, butrarely expressed GPR120. However, both GPRs were overexpressed in the L cells of the inﬂamed ileal
mucosa of CD patients. Mucosal inﬂammation induced the overexpression of GPR40, GPR120, andseveral inﬂammatory cytokines, with correlations between ileal concentrations of tumor necrosis factor(TNF)-a and GPR expression levels; however, inﬂammation did not induce the expression of proglucagon, a precursor of GLP-2 in CD patients. In rat L cells and GLUTag cells, TNF-a treatment increased
GPR120 mRNA expression without affecting GPR40 mRNA expression. Dual agonists of GPR40 and
GPR120, GW9508 and linoleic acid, respectively, increased GLP-2 production from L cells, but theseagonists decreased it in the presence of TNF-a. The GPR40 antagonist, GW1100, inhibited the GW9508induced increase in GLP-2 production, and silencing GPR120 resulted in further elevation of GLP-2
production. Thus, GPR120-dependent signaling inhibited the stimulatory effects of GPR40 on GLP-2
expression, and TNF-a treatment decreased GLP-2 expression by up-regulating GPR120 expression in L
cells. (Am J Pathol 2015, 185: 185e196; http://dx.doi.org/10.1016/j.ajpath.2014.09.010)

Crohn disease (CD) involves an abnormal immune status,
including the enhanced production of inﬂammatory cytokines,
such as ILs and tumor necrosis factor (TNF)-a. Among thesecytokines, TNF-a plays a key role in the pathogenesis of CD,
and this cytokine is a main target in CD treatment.1,2
Currently, antieTNF-a agents are widely used in CD patients and have been effective in inducing remission andmucosal healing. However, some CD patients exhibit aninsufﬁcient response or tolerance to these agents.
Glucagon-like peptides (GLPs), designated GLP-1 and
GLP-2, have been recently shown to be involved in metabolic
disorders, such as obesity and type 2 diabetes.3 GLP-1 is a
30eamino acid peptide that regulates glycemia by stimulating glucose-dependent insulin secretion and inhibiting
glucagon release and gastric emptying.4e6 In contrast, GLP-2
Copyright ª 2015 American Society for Investigative Pathology.
Published by Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ajpath.2014.09.010

is a highly conserved 33eamino acid peptide that regulates
gastric motility, gastric acid secretion, and intestinal hexose
transport.7e9 In addition to these effects, GLP-2 has antiinﬂammatory effects in the gastrointestinal tract. Experimental studies have demonstrated that GLP-2 reduces
mucosal injury and cytokine expression in several models of
colitis.10e14 Furthermore, the effects of GLP-2 on thegastrointestinal tract have been conﬁrmed by several clinical
trials. Teduglutide, a GLP-2 analogue, increases mucosal
healing and remission rates in patients with active moderate
to severe CD.15 This drug also improves intestinal functions
Supported by the Department of Gastroenterology, Osaka City University Graduate School of Medicine, Osaka, Japan.
Disclosures: None declared.

Tsukahara et al
by increasing villus height in patients with short bowel syndrome.16,17 Thus, drugs affecting GLP-2 expression and
GLP-2 itself and its analogue could be effective novel therapies against CD.
GLP-2 and GLP-1 are produced by the unique enteroendocrine L cells in the distal small intestine and colon. Both
GLP-1 and GLP-2 are simultaneously produced via posttranscriptional processing of proglucagon by prohormone
convertase 1/3 (PC 1/3) in L cells.18e20 Although mechanismsof GLP-1 secretion are well established, those of GLP-2 remain
unclear, and recent studies have suggested that different
mechanisms may exist for GLP-1 and GLP-2 secretion.21,22
G-proteinecoupled receptors (GPRs) are importantsignaling molecules for many aspects of cellular function andare members of a large receptor family that share common
structural motifs, such as seven transmembrane helices, andthe ability to activate heterotrimeric G proteins, such as Gs,

Gi, and Gq. Ligands bind speciﬁcally to GPRs to stimulate avariety of cellular responses via second messengers(eg, cAMP, Ca2þ, inositol 1,4,5-triphosphate, and diacyl
glycerol).23,24 The activation of Gs proteins results in an
increase in the cellular concentration of cAMP, leading tophosphorylation of protein kinase (PK) A, whereas the activation of Gi proteins results in a decrease in the cellularconcentration of cAMP. GPRs that are coupled to Gq proteinsuse diacyl glycerol as a second messenger. Several GPRs,such as GPR40, GPR41, GPR43, and GPR120, have beenreported to function as receptors for free fatty acids(FFAs).25e30 Some studies have demonstrated that L cells
express both GPR40 and GPR120 and secrete GLPs when
activated by medium- and long-chain FFAs, including linoleic acid (LA), suggesting that FFAs are ligands for GPR40
and GPR120 on L cells.28e30 However, the mechanisms ofregulation of GLP-2 production by these ligands and the

Figure 1 A: The expression levels of G-protein-coupled receptor (GPR) 40 and GPR120 in the ileal mucosa of patients (CD) by Western blot analysis. B:
GPR40 and GPR120 mRNA expression in the ileal mucosa of patients (CD) by TaqMan real-time RT-PCR. Protein expression and gene expression are presented aspercentages of the control. Data are presented as means  SEM in a group. C: The correlation between GPR40 and GPR120 expression levels in the ileal mucosa
of Crohn disease (CD) patients (C). D: IHC staining of GPR40 and GPR120; GPR40 (red), GPR120 (green), and DAPI (blue). Intestinal epithelial cells express
GPR40, but they rarely express GPR120, in the normal ileal mucosa. Both GPR40 and GPR120 proteins are overexpressed in the inﬂamed ileal mucosa of CDpatients. Double staining with GPR40 and GPR120 antibodies shows that these two GPRs are co-expressed on spindle-shaped cells in the intestinal glands,
which were L cells (arrows). E: The correlation between GPR40 or GPR120 expression and Harvey-Bradshaw index (HBI) of CD patients. *P < 0.05, **P < 0.01
versus control. Scale bar Z 50 mm (D).

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Roles of GPRs in Crohn Disease
ulceration, edema, and redness in the distal ileum. The patient
cohort consisted of 13 men and 3 women, with a median ageof 36 years (range, 22 to 47 years). Some patients had beenpreviously treated with TNF-a antibody therapy2 (4 of 16
patients) or an immune modulator (2 of 16 patients), but nopatients were previously treated with corticosteroids. Normal
ileal mucosa samples were obtained from patients with coloncancer who had undergone a colectomy (n Z 15). The
normal patient cohort consisted of seven men and eight
women with a median age of 64 years (range, 34 to 83 years).
Western blot analysis, real-time RT-PCR, and enzyme
immunoassay (EIA) were performed on inﬂamed and normal
mucosa samples. Our investigation was conducted according
to the Declaration of Helsinki principles. All of the patientsprovided informed consent for the use of their ileum as ahuman sample. The ethics committee of Osaka City University Hospital approved our study (protocol number 2286).

Cell Culture

Figure 2

A: Determination of tumor necrosis factor (TNF)-a, IL-1b, andIL-6 levels in the ileal mucosa of patients by enzyme immunoassay. Cytokine production is presented as cytokine levels per 100 mg of protein. Data
are presented as means  SEM in a group. The correlations between Gprotein-coupled receptor (GPR) 40 (B) or GPR120 (C) expression and thelevels of cytokines (TNF-a, IL-1b, and IL-6) in the ileal mucosa of Crohndisease patients. *P < 0.05 versus control.

interactions between GPR40 and GPR120 are currently
unclear.
Therefore, we evaluated the expression of GPR40 and
GPR120 in the ileal mucosa of patients with CD andcorrelated the expression of these GPRs with several inﬂammatory parameters. We also studied the mechanisms
underlying GLP-2 production using rat primary L cells andmouse GLUTag L cells.

Materials and Methods
Human Samples
During the 6-year period between 2004 and 2010 at Osaka
City University Hospital (Osaka, Japan), we enrolled the 16
consecutive CD patients who underwent an ileocecal resection and had small-bowel pathological conditions, such as

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We used the rat primary L cells and the mouse GLUTag L
cells.31 Rat primary L cells were puriﬁed from the ileum of
8-week-old male Sprague-Dawley rats for in vitro studies witha medium kit (Reprocell Inc., Yokohama, Japan). As apreliminary study, we characterized these cells by ﬂow cytometric analysis. These cells expressed GPR40, GPR120,
G-proteinecoupled bile acid receptor Gpbar1 (TGR5), andproglucagon, conﬁrming that these GLP-producing cells were
L cells (Supplemental Figure S1).28,30,32
Mouse GLUTag L cells were kindly provided by Tohru Hira
(University of Hokkaido, Hokkaido, Japan) and Noriyasu Hirasawa (University of Tohoku, Tohoku, Japan) and used with thepermission of Daniel J. Drucker (University of Toronto, Toronto, ON, Canada). GLUTag cells were cultured in Dulbecco’s
modiﬁed Eagle’s medium (DMEM), supplemented with 10%
fetal bovine serum (FBS) (Hyclone; Thermo Fisher Scientiﬁc Inc., Waltham, MA) and 1% penicillin/streptomycin(Gibco by Life Technologies, Carlsbad, CA).

Reagents
Recombinant rat IL-1b and IL-6 (both from R&D Systems
Inc., Minneapolis, MN) and recombinant rat and mouse
TNF-a (Shenandoah Biotechnology Inc., Warwick, PA, andLife Technologies, respectively) were used as proinﬂammatory cytokines. GW9508 (Cayman Chemical Company, Ann Arbor, MI) and LA (MP Biomedicals, Santa
Ana, CA), which are agonists for both GPR40 and GPR120,
and GW1100 (Haoyuan Chemexpress Co, Ltd, Shanghai,
China), a speciﬁc antagonist for GPR40,33 were used toevaluate the involvement of GPRs in GLP-2 production.
U73122 and H89, which are inhibitors of phospholipase C(PLC) and protein kinase A (PKA), respectively, werepurchased from Millipore (Billerica, MA). GF109203X, an
inhibitor of PKC, and cholera toxin (CTX) were supplied
by Wako Pure Chemical Industries, Ltd (Osaka, Japan).

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Figure 3

A: The expression of proglucagon in the ileal mucosa of patients (CD) by Western blot analysis. B: The mRNA expression levels of proglucagon andprohormone convertase 1/3 in the ileal mucosa of patients (CD). TaqMan real-time RT-PCR was used to examine the expression of these genes. Protein andgene expression levels are presented as percentages of the control. Data are presented as means  SEM in a group. C: IHC staining for proglucagon in the ileumof Crohn disease patients: proglucagon (red) and DAPI (blue). Proglucagon is expressed on spindle-like cells in the epithelial glands, suggesting that the
proglucagon-producing cells are L cells (arrows). Scale bar Z 50 mm (C).

Go6983, an inhibitor of PKC, and forskolin were obtained
from Sigma Chemical (St. Louis, MO). All other reagentswere of the highest purity commercially available.

ECL Plus Western blotting reagent pack; GE Healthcare,
Buckinghamshire, UK). Proteins were detected using ECLPlus detection reagent (GE Healthcare) and imaged using a
LAS-3000 analyzer (Fujiﬁlm, Tokyo, Japan).

In Vitro Study
Rat L cells (1  106 cells per well) and GLUTag L cells(5  105 cells per well) were cultured for 24 hours with orwithout proinﬂammatory cytokines in HEPES buffer (140mmol/L NaCl, 4.5 mmol/L KCl, 20 mmol/L HEPES, 1.2mmol/L CaCl2, 1.2 mmol/L MgCl2, and 0.1% bovine serum
albumin, pH 7.4). After washing, the cells were incubated for
1.5 hours to assess GLP-2 production in the absence or presenceof agonists and antagonists of GPR40 and GPR120 and thencentrifuged (200  g for 4 minutes at room temperature). In
some experiments, the L cells were treated with agonists of both
GPR40 and GPR120 and incubated with several agents thatinteract with G-proteinedependent signaling pathways with orwithout TNF-a. The supernatant and pellet were collected for
GLP-2 EIA and extraction of mRNA, respectively.

Cytokines and GLP-2 Level Assay
Levels of TNF-a, IL-1b, and IL-6 in the ileal mucosa and levelsof GLP-2 in cell culture supernatants were determined using an
EIA kit [Human TNF-a ELISA Kit (Thermo Fisher ScientiﬁcWestern Blot Analysis
Western blot analysis was performed using antibodies thatrecognize GPR40 (Abnova Corp., Taipei, Taiwan) andGPR120 (Abcam Plc, Cambridge, UK), GLP-2 and proglucagon (both from Santa Cruz Biotechnology Inc., Dallas,
TX), and phospho-PKA (Cell Signaling Technology, Danvers, MA). Brieﬂy, 20 mg of protein per well was loaded ontoa gel for Western blot analysis. SDS-PAGE was performed
using SuperSep Ace agarose gels (Wako Pure Chemical Industries, Ltd), according to molecular weight of each protein.
After SDS-PAGE, proteins were transferred to a polyvinylidene diﬂuoride membrane, which was then blocked
using 5% skim milk and incubated with a primary antibody
recognizing GPR40, GPR120, GLP-2, proglucagon, or
phospho-PKA. Finally, membranes were incubated withhorseradish peroxidaseeconjugated anti-IgG (Amersham

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Figure 4 The effects of inﬂammatory cytokines on G-protein-coupled
receptor (GPR) 40 (A) and GPR120 (B) mRNA expression in rat L cells. The L
cells were incubated with recombinant tumor necrosis factor (TNF)-a,
IL-1b, or IL-6 for 24 hours. Then, GPR40 (A) and GPR120 (B) mRNA
expression levels were determined by TaqMan real-time RT-PCR. The
expression levels are presented as percentages of the control. Data arepresented as means  SEM in a group. n Z 8. *P < 0.05 versus control.

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Roles of GPRs in Crohn Disease

Figure 5 The effect of agonists for bothG-protein-coupled receptor (GPR) 40 and GPR120,
GW9508 (A and C), and LA (B and D), on glucagonlike peptide (GLP)-2 secretion and proglucagon
mRNA expression in the absence or presence oftumor necrosis factor (TNF)-a in rat L cells. The
cells (1  106 cells per well) were cultured for 24hours with or without recombinant TNF-a, washed,
and then incubated with GW9508 and LA for 1.5
hours for GLP-2 production. The supernatant andpellet were collected for enzyme immunoassay of
GLP-2 and extraction of mRNA, respectively. TaqMan real-time RT-PCR was used to examine theexpression of proglucagon mRNA. GLP-2 production and gene expression levels are presented aspercentages of the control. Data showmeans  SEM in a group. n Z 7 to 8. *P < 0.05,**P < 0.01 versus control.

Inc.), Human IL-1b and IL-6 ELISA kits (R&D Systems Inc.),
and rat and mouse GLP-2 EIA (Yanaihara Institute Inc., Shizuoka, Japan)]. Cytokine concentrations were displayed as theamount per 100 mg of protein. GLP-2 levels were displayed aspercentages of the control.

Expression of mRNA Using Real-Time RT-PCR
Total mRNA from human samples and L cells was extractedusing the RNAqueous and RNAqueous micro kits (LifeTechnologies), respectively. Real-time RT-PCR (TaqMan; Life
Technologies) products were examined using the Fast7500
System (Applied Biosystems by Life Technologies, Carlsbad,
CA). A One Step PrimeScript RT-PCR kit (TAKARA Bio Inc.,
Shiga, Japan), along with primers and probes (Applied

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Biosystems by Life Technologies), was used to examine
GPR40, GPR120, proglucagon, and PC 1/3 mRNA expression.

IHC Staining
Ileal specimens were ﬁxed in periodate-lysine-paraformaldehyde,
embedded in optimal cutting temperature compound (TissueTek; Sakura Fine Technical Co, Tokyo, Japan), cut into sections (4 mm thick), and stained using an immunostaining
method. Brieﬂy, ileal sections were blocked with 5% normal
donkey serum (Sigma-Aldrich, St. Louis, MO) in phosphatebuffered saline. After washing, those sections were incubatedwith goat anti-human GPR40, rabbit anti-human GPR120, orgoat anti-human proglucagon primary antibodies (Santa Cruz
Biotechnology Inc.), which were diluted in Antibody Diluent

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Figure 6 G-protein-coupled receptor (GPR) 40
and GPR120 expression (A) and GLP-2 production(B) in GLUTag cells. A: GLUTag cells were incubated with recombinant tumor necrosis factor(TNF)-a for 24 hours. Then, GPR40 and GPR120mRNA expression levels were determined by TaqMan real-time RT-PCR. The expression levels arepresented as percentages of the control. B: GLUTagcells (5  105 cells per well) were cultured for 24hours with or without recombinant TNF-a, washed,
and then incubated with GW9508 for 1.5 hours toassess glucagon-like peptide (GLP)-2 production.
The supernatant was collected for enzyme immunoassay of GLP-2. GLP-2 production levels arepresented as percentages of the control. Data showmeans  SEM in a group. n Z 6 to 8. *P < 0.05,**P < 0.01, and ***P < 0.001 versus control.

with Background Reducing Components (Dako, Glostrup,
Denmark). The sections were incubated with Alexa Fluor 488
donkey anti-rabbit IgG or Alexa Fluor 594 donkey anti-goat
IgG (Molecular Probes, Life Technologies) after washing.
Finally, DAPI Fluoromount-G (Southern Biotech, Birmingham, AL) was added to the sections. We then evaluated thesections using a ﬂuorescent microscope.

Transfection of GPR120 siRNA into Rat L Cells
Silencemag (Oz Bioscience, Marseille, France) as well as
negative control and GPR120 siRNAs (both from Ambion,
Silencer Select Pre-designed siRNA; Life Technologies)
were used as transfection reagents. L cells were cultured for
3 days in DMEM containing 10% FBS and 1% penicillin/
streptomycin before transfection. Silencemag reagent was
mixed with each siRNA and incubated for 20 minutes at
37 C. The complexes were added to primary L cells in only
DMEM for 15 minutes on a magnetic plate. Finally, DMEM
containing 10% FBS and 1% penicillin/streptomycin wasadded to the cells, and the cells were cultured for 3 days.
The ﬁnal concentration of siRNA was 20 to 50 nmol/L.

Statistical Analysis
The results are expressed as means  SEM. Signiﬁcantdifferences between means were analyzed using Student’s

190

t-test and one- or two-way analysis of variance, followed by
Tukey’s post hoc test using StatView 5.0 (Hulinks Inc.,Tokyo, Japan). Signiﬁcant correlations were also analyzed
using StatView. Statistical signiﬁcance was set at P < 0.05.

Results
The Expression of GPR40 and GPR120 and the Levels of
Proinﬂammatory Cytokine in the Ileal Mucosa
GPR40 and GPR120 mRNA and protein expression levels
were detected in the normal ileal mucosa from control patients (Figure 1, A and B). The expression levels of both
GPR40 and GPR120 were signiﬁcantly increased in theinﬂamed mucosa from CD patients, compared with thosefrom control patients, at both the protein and mRNA levels,and inﬂammation had a much stronger effect on GPR120
expression than GPR40 expression (Figure 1, A and B). We
also observed a signiﬁcant correlation between GPR40 and
GPR120 expression (r Z 0.699, P Z 0.0018) (Figure 1C).
By IHC, intestinal epithelial cells expressed GPR40, butthey rarely expressed GPR120, in the normal ileal mucosa.
Both GPR40 and GPR120 proteins were overexpressed in
the inﬂamed ileal mucosa from CD patients. Double staining
with GPR40 and GPR120 antibodies showed that thesetwo GPRs were co-expressed on spindle-shaped cells in
the intestinal glands (Figure 1D), which were L cells.34

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Roles of GPRs in Crohn Disease

Figure 7 A and B: The effect of GW1100 on the
GW9508-induced increase in glucagon-like peptide(GLP)-2 production in rat L cells with or without
tumor necrosis factor (TNF)-a. C and D: The effectof G-protein-coupled receptor (GPR) 120 siRNAtransfection on GLP-2 production and GPR120mRNA expression in L cells treated with GW9508.
Transfections were performed using the magnet
transfection method. The cells (1  106 cells perwell) were cultured for 24 hours with (D) or
without (C) recombinant TNF-a, washed, and thenincubated for 1.5 hours for GLP-2 production in
the absence or presence of GW9508. GW1100 was
added to the cells 1 hour before the addition of
GW9508. The supernatant and pellet were collected
for enzyme immunoassay of GLP-2 and extractionof mRNA, respectively. TaqMan real-time RT-PCR
was used to examine the expression of GPR120
mRNA. GLP-2 production and gene expression
levels are presented as percentages of the control.
Data are presented as means  SEM in a group.
n Z 6 to 8. *P < 0.05, **P < 0.01, and***P < 0.001 versus control.

Furthermore, we determined the correlation between GPR
expression and clinical CD activity, known as the HarveyBradshaw Index (HBI). HBI values signiﬁcantly correlatedwith GPR120 expression (r Z 0.57, P Z 0.02), but not thatof GPR40 (r Z 0.432, P Z 0.096) (Figure 1E).
Next, we determined the levels of proinﬂammatory cytokines in the ileal mucosa and their correlations with GPR
expressions. TNF-a levels in CD patients were signiﬁcantly
greater than those in control patients (Figure 2A). The levelsof IL-1b and IL-6 in CD patients tended to be higher thanthose in control patients, but the differences were not statistically signiﬁcant. We analyzed the correlations between
GPR40 or GPR120 expression and proinﬂammatory cytokine production in the ileal mucosa of CD patients (Figure 2,
B and C). Both GPR40 and GPR120 expression levels
signiﬁcantly correlated with levels of TNF-a [r Z 0.832(P < 0.0001) and r Z 0.718 (P Z 0.001), respectively], but
not those of IL-1b (r Z 0.123 and r Z 0.09, respectively)
or IL-6 (r Z 0.144 and r Z 0.259, respectively).

preparation, this could not be done. Therefore, we examinedthe expression of proglucagon, a precursor of GLP-2, in theileal mucosa. Proglucagon protein expression in the ileal
mucosa of CD patients was similar to that of control patients(Figure 3A). The expression levels of proglucagon and PC 1/3
mRNAs in the ileal mucosa of CD patients also did not differ
from those of the control patients (Figure 3B). IHC staining
showed that proglucagon was expressed on spindle-like cells
in the epithelial glands, suggesting that L cells produced
proglucagon (Figure 3C).

The Expression Levels of Proglucagon Protein andmRNA and PC 1/3 mRNA in the Ileal Mucosa

The Effects of GW9508 and LA on GLP-2 Production
from Rat L Cells with or without TNF-a Treatment

We tried to detect GLP-2 protein in the ileal mucosa byWestern blot analysis. However, because GLP-2 was rapidly
degraded by dipeptidyl peptidase IV, which was in theepithelial layer during intestinal tissue sampling and protein

GW9508, at a dose of 1 mmol/L, signiﬁcantly induced GLP-2
production from L cells. However, when combined with
TNF-a treatment, GW9508 inhibited GLP-2 production in adose-dependent manner (Figure 5A). Similarly, LA, another

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The Effect of Proinﬂammatory Cytokines on GPR40 and
GPR120 mRNA Expression Levels in Rat L Cells
TNF-a at a dose of 30 ng/mL signiﬁcantly increased theexpression of GPR120 mRNA in L cells, but it did not affectthat of GPR40 mRNA, even at a 300 ng/mL dose (Figure 4,
A and B). Neither IL-1b nor IL-6 affected GPR40 and
GPR120 mRNA expression levels.

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The Effects of GW1100 and GPR120 siRNA Transfection
on GLP-2 Production from Rat L Cells Treated with GW9508
GW1100, a speciﬁc antagonist of GPR40, signiﬁcantly
inhibited the GW9508-induced increase in GLP-2 production ina dose-dependent manner (Figure 7A). This inhibitory effect of
GW1100 was stronger when combined with TNF-a treatment,with inhibitions of GW9508-induced GLP-2 production of
51% and 45% with and without TNF-a, respectively(Figure 7B). GPR120 silencing did not affect GLP-2 production
in rat L cells treated with GW9508, whereas it markedly
increased GLP-2 production in rat L cells treated with TNF-a(Figure 7, C and D), suggesting an inhibitory effect of GPR120dependent signaling and a stimulatory effect of GPR40dependent signaling on GLP-2 production.

The Effects of G-ProteineCoupled Signaling
PathwayeModulating Drugs on GLP-2 Production fromRat L Cells
The GW9508-induced increase in GLP-2 production was
inhibited by the PLC inhibitor, U73122, and the PKC

Figure 8

The effect of G-protein signaling pathwayemodulating drugs
on glucagon-like peptide (GLP)-2 production from rat L cells treated with
GW9508 in the absence (A) or presence (B) of tumor necrosis factor (TNF)-a.
The cells (1  106 cells per well) were cultured for 24 hours with or without
recombinant TNF-a and then incubated for 1.5 hours for GLP-2 production in
the absence or presence of GW9508. L cells were pre-incubated with several
agents that inhibit G-proteinedependent signaling pathways, including thephospholipase C (PLC) inhibitor, U73122, protein kinase (PK) C inhibitors,
GF109203X and Go6983, and the PKA inhibitor, H89, for 1 hour before theaddition of GW9508. The supernatant was collected for enzyme immunoassayof GLP-2. GLP-2 production levels are presented as percentages of thecontrol. Data are presented as means  SEM in a group. n Z 6 to 8.
*P < 0.05, **P < 0.01 versus vehicle.

GPR40 and GPR120 agonist,33 increased GLP-2 productionin L cells when used alone, but inhibited GLP-2 production in
the presence of TNF-a (Figure 5B). Both GW9508 and LA
increased proglucagon mRNA expression in L cells alone,whereas these agonists decreased that expression in thepresence of TNF-a (Figure 5, C and D).

GPR40 and GPR120 Expression and GLP-2 Production in
GLUTag Cells
By using GLUTag cells, we assessed the reproducibility of
TNF-a treatment on GPR expression and GLP-2 production.
TNF-a at doses of 30 and 100 ng/mL signiﬁcantly increased
GPR120 mRNA expression in GLUTag cells, whereas TNFa did not affect GPR40 mRNA expression (Figure 6A).
GW9508 induced GLP-2 production from GLUTag cells in
the absence of TNF-a in a dose-dependent manner. However,in the presence of TNF-a, GW9508 inhibited GLP-2
production in a dose-dependent manner (Figure 6B).

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Figure 9

A: The effect of cholera toxin (CTX) on glucagon-like peptide(GLP)-2 production from rat L cells in the presence of tumor necrosis factor(TNF)-a. B: The effect of forskolin on the GW9508-induced increase of GLP2 production in rat L cells. The cells (1  106 cells per well) were cultured
for 24 hours with or without recombinant TNF-a and then incubated for 1.5
hours for GLP-2 production with or without GW9508. The cells were preincubated with CTX or forskolin for 24 or 1 hour, respectively, before theaddition of GW9508. The supernatant was collected for enzyme immunoassay of GLP-2. GLP-2 production levels are presented as percentages of thecontrol. Data are presented as means  SEM in a group. n Z 6 to 8.
*P < 0.05, **P < 0.01 versus control; yP < 0.05 versus vehicle.

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Roles of GPRs in Crohn Diseaseinhibitors, GF109203X and Go6983, but not by the PKA
inhibitor, H89 (Figure 8A), suggesting that the Gq/PLC/
PKC pathway is involved in the GPR40-mediated increasein GLP-2 production. None of these inhibitors alone
affected basal GLP-2 production or GPR40 and GPR120mRNA expression (data not shown). Furthermore, these
inhibitors did not affect GPR mRNA expression in L cells
treated with GW9508 (Supplemental Figure S2).
Next, we examined the pathways involved in the inhibitionof GLP-2 production in response to GPR120 stimulation.
Because GPR120 expression was low without TNF-a treatment, these experiments were performed with TNF-a and
GW1100 treatment. GW9508-mediated inhibition of GLP-2
production was abolished by H89, a PKA inhibitor (Figure 8B).
Neither GF109203X nor Go6983 affected GLP-2 production.
In addition, GLP-2 production was signiﬁcantly inhibitedby CTX, which catalyzes the ADP-ribosylation of Gs, activates adenylate cyclase, and, as a consequence, increases theintracellular concentration of cAMP (Figure 9A). GW9508
did not inhibit GLP-2 production in L cells treated with CTX.
Furthermore, we examined phospho-PKA expression byWestern blot analysis in rat L cells transfected with GPR120
siRNA. Phospho-PKA expression was low in the absence of
GW9508, and GPR120 silencing did not affect the phosphorylation level. GW9508-mediated activation of GPR120
markedly increased PKA phosphorylation, but this increase
was inhibited in L cells transfected with GPR120 siRNA(Supplemental Figure S3). Our results strongly suggest that
the Gs/cAMP/PKA pathway is involved in the inhibition of
GLP-2 production via GPR120. Finally, we examined theeffect of forskolin, which elevates cAMP, on L cells.

Figure 10 Regulation of glucagon-like peptide (GLP)-2 secretion
through G-protein-coupled receptor (GPR) 40, GPR120, and tumor necrosis
factor (TNF)-a under physiological and inﬂammatory conditions.

The American Journal of Pathology - ajp.amjpathol.org

Expectedly, forskolin inhibited the GW9508-induced increase in GLP-2 production (Figure 9B).

Discussion
In this study, we demonstrated that GPR40 and GPR120
expression levels were increased in the ileum of CD patients, and these GPR expression levels strongly correlatedwith intestinal TNF-a levels. However, this increase in GPR
expression levels did not contribute to the increase in the
production of proglucagon, a precursor of GLP-2, which isconsistent with the ﬁndings of a previous report that GLP-2
expression levels in the ileal and colonic mucosa weresimilar between control patients and patients with inﬂammatory bowel disease (IBD).35 These results suggest that
inhibitory signals of GPR-dependent GLP-2 production
may exist in inﬂamed intestinal tissue. A schematic representation of the pathway with regard to our results ispresented in Figure 10.
Our in vitro studies clearly demonstrated an inhibitory
effect of GPR120-dependent signaling and a stimulatory
effect of GPR40-dependent signaling on GLP-2 productionin L cells. Thus, GPR120 expression may be associated withenhanced mucosal inﬂammation and damage (Figure 10).
This is supported by the ﬁnding that GPR120 expression
levels strongly correlated with HBI scores of CD activity.
To the best of our knowledge, this is the ﬁrst report todemonstrate the role of GPR120 in GLP-2 production in an
inﬂammatory condition.
TNF-a plays important roles in the pathogenesis of CD.
This cytokine exerts pleiotropic effects in the intestinal mucosa, leading to the induction and enhancement of inﬂammation and tissue damage. TNF-a increases neutrophil adhesion
to vascular endothelial cells and promotes neutrophil transmigration through the endothelium.36,37 TNF-a also stimulates
type 1 T-helper responses38 and activates endogenous matrix
metalloproteinases, which damage the mucosa and induce
subsequent ﬁbrosis.39 Herein, the overexpression of GPR40
and GPR120 correlated with levels of TNF-a in the ileal
mucosa of CD patients. However, we found that TNF-astimulated the expression of GPR120, but not that of GPR40,
in both rat and mouse L cells. Our results suggest that although
TNF-a is a potent inducer of GPR120 expression, it does nothave a direct effect on GPR40 expression. Recently, several
factors, such as peroxisome proliferator-activated receptor-gagonists and bacteria-produced FFAs, have been reported to
up-regulate GPR expression.40,41 Although the mechanisms ofregulation of GPR40 expression are still unclear, some mediators associated with intestinal inﬂammation other thanproinﬂammatory cytokines (ie, TNF-a, IL-1b, and IL-6) may
enhance GPR40 expression in CD patients.
Of interest, although the expression levels of GPR40 and
GPR120 were increased at both the mRNA and protein
levels in CD patients, the extent of the increase in proteins
was much greater than that of the mRNAs. Although the193

Tsukahara et al
reason for this discrepancy is unclear, post-transcriptional
regulation of GPR40 and GPR120 may exist.
GLP-1 and GLP-2 are simultaneously produced by the
post-transcriptional processing of proglucagon.18e20 BothGPR40 and GPR120 have been reported to induce GLP-1
production in several cell types, including L cells andpancreatic b-islet cells. Because the dynamics of GLP-2
production have been similar to those of GLP-1 production
in many studies, these two GPRs were thought to stimulate
GLP-2 secretion. However, some reports have suggested that
GLP-2 is not released in parallel with GLP-1 in rats,21,22
suggesting that different mechanisms may exist for thesecretion of GLP-1 and GLP-2. Our ﬁndings that the GPR40dependent pathway increased GLP-2 production from L
cells, whereas the GPR120-dependent pathway decreased it,support our hypothesis.
In the absence of TNF-a, GPR120 expression was low.
Therefore, dual agonists of GPR40 and GPR120 mainly
activated GPR40. Because the effect of GW9508 on GLP-2
production in the absence of TNF-a was abolished by the
inhibition of the PLC/PKC signaling, we concluded thatGPR40 in L cells couples to Gq proteins. On the other hand,
H89, a PKA inhibitor, reversed the GPR120-induced inhibition of GLP-2 production, and GPR120 activation had noeffect in L cells treated with CTX, which suggests thatGPR120 in L cells couples to Gs proteins and activates the
PKA/cAMP pathway. Contrary to our ﬁndings, both GPR40
and GPR120 have been shown to couple to Gq protein instudies.30,42e44 Recently, the anti-inﬂammatory effects of
GPR120 activation have been shown to depend on b-arrestin2 in macrophages, independently of Gq proteins,45 implyingthat GPR120 uses other G proteins in macrophages. Thus,
GPR120-coupled G proteins may differ in different cells.
Several lines of evidence suggest that PKA signaling
antagonizes PLC/PKC signaling.46e48 Consistently, in thisstudy, PKA signaling inhibited PLC/PKC-induced GLP-2
production in L cells. Together with the ﬁnding that forskolin,which elevates cellular concentrations of cAMP, inhibited the
GPR40/Gq-induced increase in GLP-2 production, theseresults suggest that these PKA-PKC interactions may occur inthe signaling systems of L cells.
Many experimental and clinical studies have demonstrated the efﬁcacy of GLP-2 analogues in the treatment ofcolitis, which suggests that GPR40 agonists would be also
effective in the treatment for IBD, especially CD. However,
our results indicate that these agonists may be ineffective forintestinal inﬂammation patients, in whom TNF-a induces
GPR120 expression. Although no clinical trials to evaluate
the efﬁcacy of GPR40 agonists in IBD have been reported,
epidemiological studies have demonstrated that dietary fat
enriched in n-6 fatty acids, which mainly consist of LA,
was an increased risk factor, rather than a protective
factor, for the incidences of CD and ulcerative colitis.49,50
GPR120 antagonists or combination therapies of GPR40 agonists and antieTNF-a agents, which would decrease GPR120
expression, could be novel candidates for IBD therapy.

194

In conclusion, GPR120-dependent signaling inhibitedthe stimulatory effects of GPR40-dependent signaling on
GLP-2 expression, and TNF-a decreased GLP-2 expression by up-regulating GPR120 in L cells, leading to theaggravation of inﬂammation and disturbance of woundhealing in CD. Clinical data showing an antieTNF-aagenteinduced increase in GLP-2 production would
strengthen our hypothesis.

Acknowledgments
We thank Daniel J. Drucker (University of Toronto, Toronto, ON, Canada) for permission to use the GLUTag L
cells and Tohru Hira (University of Hokkaido, Hokkaido,
Japan) and Noriyasu Hirasawa (University of Tohoku,
Japan) for providing the GLUTag L cells.

Supplemental Data
Supplemental material for this article can be found at
http://dx.doi.org/10.1016/j.ajpath.2014.09.010.

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